Ribonucleic acid (RNA) molecules have gained much attention over the last years, particularly in the medical field. Thus, therapeutic RNA molecules represent a promising class of drugs, wherein RNA-based therapeutics include mRNA molecules encoding antigens for use as vaccines (Fotin-Mleczek et al. (2012) J. Gene Med. 14(6): 428-439). In addition, it is envisioned to use RNA molecules for replacement therapies, e.g. by providing missing proteins such as growth factors or enzymes to patients (Karikó et al. (2012) Mol. Ther. 20(5):948-953; Kormann et al. (2012) Nat. Biotechnol. 29(2):154-157, Thess et al. (2015) Mol. Ther. doi: 10.1038/mt.2015.103). Furthermore, the therapeutic use of noncoding immunostimulatory RNA molecules (Heidenreich et al. (2014) Int J Cancer. December 21. doi: 10.1002/ijc.29402) and other non-coding RNAs such as microRNAs and long noncoding RNAs is considered (Esteller (2011) Nat. Rev. Genet. 15 12(12):861-74).
In view of the above, there is a permanent need for new methods of producing RNA, which result in a high yield of the RNA to be produced. Furthermore, the RNA has to be produced in a high quality, particularly if it is applied in the medical field. Cost-efficacy of such new methods is of course also an important factor. Accordingly, new methods are sought after in the field of RNA production, which on the one hand result in RNA in the desired yield and quality, and on the other hand are economical in terms of the number of steps, the complexity of the steps, and the resources used in the steps.
WO 2012/170433 in the name of Sutro Biopharma, Inc. discloses means to transcribe circular plasmid DNA into either tRNA or mRNA. The method disclosed therein involves the use of multiple DNA:RNA polymerase termination sequences to prevent waste of high energy phosphates due to inefficient termination of the polymerases, wherein the focus in WO 2012/170433 is on polymerase termination sequences (or elements) comprising at least one Class I termination sequence in combination with at least one Class II termination sequence. WO 2012/170433 is in particular concerned with the production of tRNA, see claim 1 of WO 2012/170433.
WO 2014/186334 in the name of Robert Kruse discloses a method of producing a circular RNA, preferably circular mRNA, wherein the underlying DNA vector comprises two elements flanking the sequence of interest, namely a self-circularizing intron 5′ slice junction and a self-circularizing intron 3′ slice junction resulting in self-circularization of the RNA after transcription. The DNA vector may optionally contain an RNA polymerase terminator sequence.